Jockovic Et Al., 2008_JSCS-3795

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ISSN 0352–5139UDC 54:66

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J. Serb. Chem. Soc. 73 (12) 1161–1167 (2008) UDC 54+582.33:547.261:547.56

JSCS–3795 Original scientific paper

doi: 10.2298/JSC0812161J1161

HPLC–DAD of phenolics in bryophytes Lunularia cruciata, Brachytheciastrum velutinum and Kindbergia praelonga

NEBOJŠA JOCKOVIĆ1, PAULA B. ANDRADE2, PATRÍCIA VALENTÃO2

and MARKO SABOVLJEVIĆ3

* 1 Institute of Pharmaceutical Biology, Martin-Luther-University Halle-Wittenberg,

Hoher Weg 8, 06120 Halle/Saale, Germany, 2 Requimte, Institute of Pharmacognosy,

Faculty of Pharmacy, University of Oporto, Rua Aníbal Cunha, 4050-047, Porto,

Portugal and 3 Institute of Botany and Garden, Faculty of Biology, University of

Belgrade, Takovska 43, 11000 Belgrade, Serbia

(Received 27 March, revised 30 May 2008)

Abstract : The chemistry of bryophytes is not well known. The available dataindicate interesting chemical constitutions of some bryophyte species, i.e., ac-tive and new compounds are to be found within bryophytes, especially liver-worts. In this study, one liverwort and two moss species were studied: Lunu-

laria cruciata (L.) Dumort, Brachytheciastrum velutinum (Hedw) Ignatov &

Huttunen and Kindbergia praelonga (Hedw) Ochyra. The phenolic compo-sitions of these bryophyte species have not hitherto been reported. Their metha-nolic extracts were analyzed by reversed-phase HPLC, coupled to a diode-arraydetector (DAD). Luteolin-7-O-glucoside and quercetin were found in the L.

cruciata extract. The extract obtained from B. velutinum contained four phe-nolic acids (4-O-caffeoylquinic, 5-O-caffeoylquinic, caffeic and ellagic acids)and three flavonoids (apigenin-7-O-glucoside, luteolin and apigenin). The K.

praelonga extract was characterized by the presence of several phenolic acidsand their derivatives (4-O-caffeoylquinic, 5-O-caffeoylquinic, caffeic, p-cou-maric, ferulic and ellagic acids, and caffeic and p-coumaric acid derivatives)and three flavonoids (apigenin-7-O-glucoside, luteolin, apigenin and an un-identified flavanone).

Keywords: bryophytes; phenolics; Lunularia cruciata; Brachytheciastrum velu-

tinum; Kindbergia praelonga.

INTRODUCTION

Bryophytes (mosses, liverworts and hornworts) with approximately 15,000– –25,000 species1 are, after flowering plants, worldwide the most diverse plantgroup. They are to be found in all ecosystems, from desert to alpine, except ma-rine, and the bryophyte biomass productivities can vary in each ecosystem, from

* Corresponding author. E-mail: [email protected]

Author's personal copy

(c) 2008 Serbian Chemical Society.

Available on-line at www.shd.org.rs/JSCS/



1162 JOCKOVIĆ et al .

negligible to the most significant producers. However, the ecological role of bryophytes in any ecosystems is significant.

The chemistry of bryophytes is poorly known and the results on are veryscattered.2–4 The reason for this is the difficulty in identification and smallamount of the same species available for analyses, usually by sophisticated me-thods. Liverworts are very interesting for chemical analysis due to their oil bo-dies containing many scientifically new compounds.

However, worldwide bryophytes are known to be used in ethno-botany andare applied to cure diseases, threat to plants and animals, or in the household.5–7 Therefore, bryophytes are indicated as a source of chemically new and unknowncompounds.3,4,8–10 Studies of the chemical constituents of bryophytes were re-cently performed but are still inadequate and neglected.2,11–18 These data help inthe systematics of barely morphologically classified bryophytes.19 Also, somescattered data on the biological activities of bryophyte extracts and/or chemicalconstituents are available for not very many bryophyte taxa.20–23

Generally, based on the species studied to date, bryophytes are known to possess extremely high amounts of terpenoids, phenolics (flavonoids and biben-zyl derivatives), glycosides, fatty acids and also some rare aromatic compounds.Bryophytes are considered as a “remarkable reservoir” of new, natural productsor secondary compounds, many of which have shown interesting biological acti-vity. These activities of bryophytes include antimicrobial, antifungal, cytotoxic,

antitumor, vasopressin (VP) antagonist, cardiotonic, allergy causing, irritancyand tumour effecting, insect anti-feedant, insecticidal, molluscicidal, pesticidal,

plant growth regulatory, superoxide anion radical release inhibition and 5-lipo-xygenase, calmodulin, hyaluronidase and cyclooxygenase inhibition features.Some latest results also predict a beneficial influence of bryophytes in AIDStherapy (some bibenzyls of liverworts).24–36

The liverwort Lunularia cruciata, a Mediterranean Atlantic species, expres-ses antimicrobial and, to a less extent, antifungal activities.37–39 The plant--growth-regulator lunularic acid was isolated for the first time from this spe-cies.40 The chemical constituents of L. cruciata are unknown.4,41

The palearctic mosses Brachytheciastrum velutinum and Kindbergia prae-

longa have hitherto not been chemically screened; nor are their bioactive effectsknown.4

EXPERIMENTAL

Samples

Fresh material was collected in July 2003 in the Oporto City Park (Portugal). A voucher of each Bryophyte sample is deposited in the Bryophyte Collection of Belgrade University(BEOU).

The material was cleaned and dried to constant weight at room temperature.





HPLC–DAD OF PHENOLICS IN BRYOPHYTES 1163

Extraction of phenolics

5 g dry mass of each bryophyte sample was used for the extraction of the phenolics. Thematerial was previously ground in an electric mill to a rough powder. The extraction consistedof two consecutive steps employing 175 and 125 mL methanol, respectively, on a magneticstirrer for 10 min. These two extracts were combined and the solvent removed under reduced

pressure at 30 °C To this residue, 20 mL of 2.0 M HCl were added and the obtained solutionwas passed through a C18 Bond Elut cartridge, preconditioned with methanol and 2.0 M HCl.The retained phenolics were eluted with methanol. This solution was taken to dryness under reduced pressure (30 °C), dissolved in methanol and 20 µL were analyzed by HPLC–DAD.

HPLC–DAD analysis of the phenolics

The extracts were analyzed on an analytical HPLC instrument (Gilson), using a Sphe-risorb ODS2 column (25.0 cm0.46 cm; 5 µm particle size Waters, Milford, MA, USA) witha C18 ODS guard column. The mobile phase consisted of solvent A (water–formic acid(19:1)) and solvent B (methanol) (Table I).

The flow rate was 0.9 mL/min and the injection volume 20 μL. Detection was performedusing a Gilson diode array detector. The phenolic compounds in each sample were identified

by comparing their retention times and UV–Vis spectra in the 200–600 nm range with in-dividual standards. The chromatograms were registered at 280, 320 and 350 nm.

TABLE I. Gradient flow

Time, min Solvent A content, % Solvent B content, %0.00 95 53.00 85 15

13.00 75 25

25.00 70 3035.00 65 3539.00 60 4042.00 55 4544.00 50 5047.00 45 5550.00 30 7056.00 25 7560.00 0 10062.00 5 95

RESULTS AND DISCUSSION

The chromatogram of the methanol extract of Lunularia cruciata is pre-

sented in Fig. 1. Based on a comparison of the retention time ( Rt ) and UV–Visspectra with standard substances, the presence of the flavonoid heteroside luteo-lin-7-O-glucoside and the flavonoid aglycone quercetin was confirmed. The pre-sence of these two compounds is for the first time reported in L. cruciata.

The chromatogram of the methanol extract of Brachytheciastrum velutinum is presented in Fig. 2. The following substances were evidenced as constituentsof this species: phenolic acids, i.e., 4-O-caffeoylquinic, 5-O-caffeoylquinic, caf-feic and ellagic acid, flavonoids, i.e., heteroside apigenin-7-O-glucoside, and fla-vonoid aglycones, i.e., luteolin and apigenin.






0 20 40 60

0.0

0.2

21

I n t e n s i t y , a . u .

/ min

Fig. 1. Chromatograms of the methanol extractof liverwort L. cruciata: luteolin-7-O-

-glucoside (1) and quercetin (2).

0 20 40 60

0.0

0.2

7

6

54

321


/ min Fig. 2. Chromatogram of the moss methanolextract of B. velutinum: 4-O-caffeoylquinic

acid (1), 5-O-caffeoylquinic acid (2), caffeicacid (3) and ellagic acid (5), apigenin-7-O-

glucoside (4), luteolin (6) and apigenine (7).

0 20 40 60

0.0

0.2

**

*

9

87

65

4

321


/ min

Fig. 3. Chromatogram of the methanol extractof the moss K. praelonga: 4-O-caffeoylquinicacid (1), 5-O-caffeoylquinic acid (2), caffeicacid (3), p-coumaric acid (4), ferulic acid (5),ellagic acid (7), caffeic acid derivative (*),

p-coumaric acid derivative (**), apigenin-7-O--glucoside (6), luteolin (8), apigenin (9) and un-identified flavanone ().

In the methanol extract of the moss Kindbergia praelonga, 4-O-caffeoylqui-nic, 5-O-caffeoylquinic, caffeic, p-coumaric, ferulic and ellagic acid, caffeic acidderivative, p-coumaric acid derivative, flavonoid heteroside apigenin-7-O-gluco-side, aglycones luteolin and apigenin, as well as one unidentified flavanone wereevidenced, as shown in Fig. 3.

The chemical contents of B. velutinum and K. praelonga have not beenscreened previously.






Luteolin is present in many vascular plants, especially from the family Rese-daceae, Genista tinctoria (Fabaceae) and Petroselinum crispum (Apiaceae).42 How-ever, the heteroside form of luteolin-7-O-glucoside is not common and this com-

pound was not previously known from L. cruciata. This form is known fromsome Mentha plants.43 The yellowish pigment quercetin is widespread in many

plants but was not detected previously in L. cruciata. Quercetin was found to bethe most biologically active of the flavonoids and many medicinal plants owemuch of their activity to their high quercetin content.44

Artichoke (Cynara scolymus) is known to have rich content of 4-O-caffeoyl-quinic and 5-O-caffeoylquinic acids.45 Previously they were not evidenced frommosses among the other phenolic acids.4

Caffeic acid is already known from some mosses.46 Apigenine is a pale yel-low pigment present in many plants from the families Apiaceae and Asteraceaewith an antitumor effect. Apigenin and its derivates are known to be present inmosses and to have biological effects.47 In mosses, p-coumaric and ferulic acidsare known to be present in moss spores. They are precursors of lignin, which isnot common in moss gametophytes, but both p-coumaric and ferulic are presentin moss gametophytes where lignin was not detected.48

Although phenolic compounds are known to be present in bryophytes, thisknowledge is mainly based on liverworts not mosses and their presence; diversityand distribution within different species remain for further studies.49–51

Thus, the paper presents one first approach to the identification of phenolicsin the bryophytes L. cruciata, B. velutinum and K. praelonga, until now unknown.

Acknowledgements. M. Sabovljević thanks the Serbian Ministry of Science for support(Grant No. 143015).

И З В О Д

HPLC–DAD ФЕНОЛА КОД БРИОФИТА Lunularia cruciata, Brachytheciastrum velutinum И Kindbergia praelonga

NЕБОЈША JОЦКОВИЋ1, PAULA B. ANDRADE2, PATRÍCIA VALENTÃO2 и MАРКО САБОВЉЕВИЋ3

1 Institute of Pharmaceutical Biology, Martin-Luther-University Halle-Wittenberg, Hoher Weg 8,

06120 Halle/Saale, Germany, 2 Requimte, Institute of Pharmacognosy, Faculty of Pharmacy,

University of Oporto, Rua Aníbal Cunha, 4050-047, Porto, Portugal и 3Institut za botaniku

i botani~ka ba{ta,Biolo{ki fakultet, Univerzitet u Beogradu, Takovska 43, 11000 Beograd

Хемијски састав бриофита је слабо познат. Dосадашњи подаци указују на интересантне хемијске састојке бриофита, биолошки активна и нова једињења, нарочито код јетрењача. У овом раду изучаване су једна јетрењача Lunularia cruciata (L.) Dumort и две маховине Bra-

chytheciastrum velutinum (Hedw) Ignatov & Huttunen и Kindbergia praelonga (Hedw) Ochyra.Фенолни састав ових врста бриофита од раније није познат. Њихови метанолни екстракти су анализирани путем HPLC типа реверсне фазе, повезаног са DAD детектором. U екстракту L.

cruciata пронађени су лутеолин-7-O-глукозид и кверцетин. Eкстракт добијен од B. velutinum показао је присуство четири фенолне киселине (4-O-кафеоилхина, 5-O-кафеоилхина, кофе-инска и елагинска киселина) и три флавоноида (флавоноидни агликони лутеолин и апиге-






нин, и његов хетерозид апигенин-7-O-глукозид). Eкстракт од K. praelonga је окарактерисан присуством неколико фенолних киселина и њихових деривата (4-O-кафеоилхина, 5-O-ка-феоилхина, кофеинска, н-кумаринска, ферула и елагинска киселина, деривати кофеинске и н-кумаринске киселине) и следећих флавоноида: апигенина, апигенин-7-O-глукозида, лутео-лина и једног неидентификованог флаванона.

(Примљено 27. марта, ревидирано 30. маја 2008)

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